EP2865748A1 - Milieu de culture pour bactéries du genre clostridium exempt de constituants d'origine animal et procédé de production d'un surnageant contenant une ou plusieurs protéases à activité collagénolytique et gélatinolytique - Google Patents

Milieu de culture pour bactéries du genre clostridium exempt de constituants d'origine animal et procédé de production d'un surnageant contenant une ou plusieurs protéases à activité collagénolytique et gélatinolytique Download PDF

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EP2865748A1
EP2865748A1 EP13796869.9A EP13796869A EP2865748A1 EP 2865748 A1 EP2865748 A1 EP 2865748A1 EP 13796869 A EP13796869 A EP 13796869A EP 2865748 A1 EP2865748 A1 EP 2865748A1
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culture medium
culture
histolyticum
supernatant
collagenolytic
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EP2865748A4 (fr
EP2865748B1 (fr
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Marcos Castanheira Alegria
Lucidio Cristóvão FARDELONE
Marina Baiochi Riboldi DELALANA
Josef Ernst Thiemann
Spartaco Astolfi Filho
Roberto Carlos Debom Moreira
Ogari De Castro Pacheco
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CRISTALIA PRODUTOS QUIMICOS FARMAC UTICOS Ltda
Cristalia Produtos Quimicos e Farmaceuticos Ltda
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CRISTALIA PRODUTOS QUIMICOS FARMAC UTICOS Ltda
Cristalia Produtos Quimicos e Farmaceuticos Ltda
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24003—Microbial collagenase (3.4.24.3)

Definitions

  • the present invention is related to the field of microbiology and biotechnology. Particularly, this invention is related to a culture medium for bacteria of the genus Clostridium, animal product-free, and a process for producing supernatant comprising one or more protease with collagenolytic and gelatinolytic activity.
  • Collagenase (clostridiopeptidase A, EC 3.4.24.3) is a proteolytic enzyme capable of hydrolyzing collagen proteins, both in their native and denatured (gelatins) form. No other enzyme is capable of cleaving native collagen, due to its distinct amino acid composition (presence of a numerous amount of imino acids, represented by tripeptide Gly-Pro-X, where X is, frequently, proline or hydroxyproline), and its single macrostructure (complex triple-helix structure).
  • Collagenase isolated or as the main component of compositions comprising other proteolytic enzymes, has been widely used since the 70's in the treatment of several diseases and pathological conditions. All diseases and conditions in which collagenase is administered are associated with the excessive deposit of collagen and the erratic accumulation of fibrous tissue rich in collagen. Such diseases and conditions are denominated collagen-mediated diseases, and the local application of compositions containing collagenase is one of the possible treatments serving as an alternative for surgical intervention. The use of compositions containing collagenase for the treatment of necrotic tissue debridement (burns, skin lesions, etc.) has also been approved by regulatory agencies, in which such administration has shown surprising results on improving scarification processes.
  • compositions containing collagenase are: burn treatment, with a concomitant beneficial effect over tissue regeneration; enzymatic debridement of wounds; infected wounds and ulcer treatment (of skins and decubitus ulcers); intervertebral disc hernia treatment; selective lysis of collagen fibers of the eye's vitreous body; Dupuytren's disease treatment; Peyronie's disease treatment; adhesive capsulitis treatment; lateral epicondylitis treatment, Carpal Tunnel Syndrome and plantar fasciitis; increase of regeneration of damaged nerves; also being widely used in medical specialties such as plastic surgery and in several dermatological and aesthetic procedures, for example, in the treatment of cellulites and mammal scars, such as acne, keloid and other hypertrophic scars through intralesional injection of purified collagenase.
  • compositions containing collagenase in rheumatoid arthritis, metastasis, angiogenesis and cirrhosis.
  • Collagenases have also been described as important tools in specific organ transplantation i.e. isolating of pancreatic islets of Langerhans, microvascular endothelial cells, hepatocytes and chondrocytes ( WO9824889 ). Tissue dissociation mediated by collagenolytic enzyme is a critical step in several cell isolation procedures.
  • the collagenases used in the majority of the therapeutic applications described above are extracted from the supernatant culture, purified or not, of bacterial cultures, most specifically of Clostridium histolyticum cultures.
  • Clostridium histolyticum is an anaerobic, gram-positive and spore-forming cylindrical bacterium.
  • the collagenolytic activity of the extracellular enzymes of such bacteria has been known for over 50 years, when it was isolated for the first time an extracellular enzyme capable of digesting cattle Achilles tendons.
  • Clostridium sp most specifically Clostridium histolyticum, is still the main source of collagenolytic and gelatinolytic enzymes for therapeutic application due to its higher enzymatic productivity and activity against several collagen forms and its peptides.
  • collagenase sources for therapeutic use include mammal cells, crustaceans (crabs, shrimp), fungi and other bacteria (Streptomyces, Pseudomonas and Vibrio).
  • Clostridium collagenases to digest several kinds of collagen (type I, II, III, VII and X) and gelatin is the major factor that differentiates these proteases from other collagenase sources.
  • Aforesaid clostridial enzymes not only present higher collagenolytic activity when compared to vertebrate collagenases but it also presents an optimum pH for its activity within human physiological pH range.
  • collagenases classes I and II
  • other important proteases are also present, some such are: neutral proteases i.e.: gelatinases, clostripains (clostridiopeptidase B, EC 3.4.22.8), trypsins, elastases and aminopeptidases.
  • Non-proteolytic enzymes including: galactosidase, acetylglucosaminidase, fucosidase, phospholipase, neuraminidase (or sialidase) and hyaluronidase can also be found.
  • galactosidase acetylglucosaminidase
  • fucosidase phospholipase
  • neuraminidase or sialidase
  • hyaluronidase hyaluronidase
  • collagenase are deeply involved in the degradation process of extracellular matrix components. The presence of this "enzymatic combination" is required to obtain compositions suitable to be used as enzymatic debridement agent for skin lesion and organ transplant (i.e. isolation of pancreatic islets).
  • collagenase composition for tissue dissociation and debridement is a crude or partially purified preparation (obtained by few purification steps). Since the presence of enzymes which act on native collagen and reticular fibers in addition to enzymes responsible for the hydrolysis of other proteins, polysaccharides and lipids in the extracellular matrix of connective and epithelial tissue are required. Although such preparation does not require complex purification steps, the difficulty lies in the production process, due to the different expression patterns of the proteolytic enzymes required in the final composition.
  • compositions of collagenase for the treatment of collagen-mediated diseases require high purity of the preparations, due to the need of a specific action on digestion of collagen; such treatments are administered through local injections.
  • processes of purification of the supernatant of C. histolyticum cultures are very long, including numerous steps with different strategies of purification.
  • the collagenase composition purity is directly related to the therapeutic application intent.
  • a defined process for culturing Clostridium histolyticum is essential to direct the production in order to obtain the proteases of interest in the supernatant, purified or not.
  • the enzymatic activity and the concentration of proteases in C. histolyticum cultures supernatant vary according to the bacterial strain, culture medium, culture conditions, culture age, cellular density, among other variables.
  • the intrinsic variability of this type of biotechnological process implies in exhaustive quality control analysis of the produced batches, increasing production costs.
  • the loss of efficacy of traditional collagenase along time, regardless of the storage conditions is strongly related to the enzymatic composition of the supernatant. For such reason, defined culture conditions which direct the production of the desired enzymes collagenolytic and gelatinolytic enzymes, in special collagenases are required.
  • peptones Commonly described as examples of animal origin peptones are: bacto-peptone, peptone proteose, hydrolyzed casein, casein tryptic hydrolysate, among others. Combinations of these components with vegetable peptones have also been described.
  • Clostridium histolyticum is considered capable of fermenting amino acids, and is sub-classified in the so called Group II (C. botulinum; C. histolyticum; C. cochlearium; C. subterminale ).
  • Group II C. botulinum; C. histolyticum; C. cochlearium; C. subterminale .
  • serine, glutamine, glycine and valine are the main amino acids used by these species, the addition of theses in culture medium produces no or little effect on the bacterial growth.
  • C. histolyticum tolerates few amounts of oxygen, characteristic common in some anaerobes and also other species of the genus, for such reason it can be considered an aero tolerant microorganism. Therefore, minimal conditions of anaerobiosis must be secured for culturing C. histolyticum for protease production with high collagenolytic and gelatinolytic activity.
  • reducing agents are also considered critital in anaerobe liquid culture.
  • reducing agents are also considered critital in anaerobe liquid culture.
  • the most frequently used agents are: digest of meat, glucose, sodium thioglycolate, cysteine, iron salts, metallic iron or a mixture of both.
  • the conventional C. histolyticum culturing processes present several disadvantages, such as: low yield, low reproducibility and incomplete separation of impurities. Furthermore, one of the major problems such process is the predominant use of animal origin components in the production of proteases with collagenolytic and gelatinolytic activity for therapeutic use in humans.
  • soybean and yeast extracts can be used to replace animal derived peptones in some species of the genus Clostridium such as C. sporogenes, C. tetanus, C. botulinicum.
  • Such extracts are capable of sustaining growth rate as well as protease expression and secretion
  • the culture media described so far for the C. histolyticum presents disadvantages such as: presence of animal components, high cost associated with its complex composition, low collagenase expression and long culturing periods required to obtain a supernatant with collagenolytic and gelatinolytic activity appropriate for industrial production with therapeutic purposes.
  • WO2007089851 (2007; Auxilium ) describes C. histolyticum culture conditions in order to obtain collagenases. Although it describes the production of collagenases by C. histolyticum in an animal product-free culture medium, it still emphasizes that in order to produce collagenases with higher reproducibility, a culture medium comprising peptone protease of animal origin is preferred. Only the animal origin culture medium was capable of producing the collagenases in adequate proportion to maximize its synergistic activity, resulting in a therapeutic benefit.
  • the culture medium described above may contain amino acids (glutamine, tryptophan and asparagine), yeast extract, inorganic salts, glucose and vitamins.
  • the present invention provides a culture medium for bacteria of the genus Clostridium, preferably C. histolyticum, animal product-free and a process for the production of supernatant comprising one or more proteases with collagenolytic and gelatinolytic activity adequate for industrial collagenolytic and gelatinolytic enzymes production, in particular collagenases, for therapeutic purposes.
  • the present invention refers to an animal product-free culture medium for bacteria of the genus Clostridium comprising water, non-animal origin peptone or its derivatives, yeast extract and the amino acids cysteine and arginine, or pharmaceutically acceptable salts thereof.
  • the bacteria belonging to Clostridium histolyticum strain Preferably, the bacteria belonging to Clostridium histolyticum strain.
  • the culture medium of the present invention can additionally comprises one or more additives selected from the group consisting of agent for adjustment pH in the range between 7 and 8, reducing agent, inorganic salts and vitamins.
  • the non-animal origin peptone may be vegetable or yeast peptone.
  • the non-animal origin peptone is a vegetable peptone selected from the group consisting of soybean, cotton, wheat, sunflower, rice, peanut, fava bean, peas, potato, corn or mixtures thereof.
  • the vegetable peptone is a soybean peptone.
  • the culture medium of the present invention is characterized by the fact that the non-animal origin peptone concentration ranges from 0.5% to 5% w/v; yeast extract concentration ranges from 0.5% to 5% w/v; cysteine concentration, or its pharmaceutically acceptable salts, ranges from 0.01% to 0.1% w/v; and arginine concentration, or its pharmaceutically acceptable salts, ranges from 0.1% to 1% w/v.
  • the non-animal origin peptone concentration ranges from 1% to 4% w/v; the yeast extract concentration ranges from 1% to 4% w/v; the cysteine concentration, or its pharmaceutically acceptable salts, ranges from 0.03% to 0.07% w/v; and the arginine concentration, or its pharmaceutically acceptable salts, ranges from 0.25% to 0.7% w/v.
  • the non-animal origin peptone concentration is 3% w/v; the yeast extract concentration is 3% w/v; the cysteine concentration, or its pharmaceutically acceptable salts, is 0.0625% w/v; and the arginine concentration, or its pharmaceutically acceptable salts, is 0.375% w/v.
  • the culture media of the present invention are animal product-free and, surprisingly, stimulate proteases with collagenolytic and gelatinolytic activity production, particularly collagenases, being, such as it is, superior to the conventional culture media which contain animal origin peptones.
  • the present invention also refers to a process for Clostridium histolyticum liquid culturing in order to produce a supernatant comprising one or more proteases with collagenolytic and gelatinolytic activity characterized by comprising the steps of: a) providing a sterile animal product-free culture medium; b) culturing an aliquot of Clostridium histolyticum stock culture in the culture medium in step (a), under anaerobic conditions at about 37°C before reaching the stationary growth phase in order to obtain an inoculum; c) providing a sterile animal product-free culture medium for Clostridium bacteria, according to present invention; d) adding to the culture medium in step (c) a volume of inoculum from step (b) equal to or lower than 10% of the final volume of the medium defined in (c); e) culturing the C.
  • step (f) removing the cellular material and other particulate matter in the liquid phase of the culture in step (e), producing a supernatant comprising one or more proteases with collagenolytic and gelatinolytic activity.
  • Such process may include one or more additional steps for the purification of one or more proteases with collagenolytic and gelatinolytic activity present in the supernatant obtained in step (f).
  • the present invention also refers to a supernatant of a C. histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activity obtained according to the process mentioned above, and pharmaceutical compositions comprising, as active ingredient, the supernatant of Clostridium histolyticum liquid culture and pharmaceutically acceptable excipients.
  • Figure 1 Comparison between the bacterial growth (optical density 600 nm) of C . histolyticum (ATCC 21000) in an animal product-free culture medium (3% w/v of vegetable peptone, 3% w/v of yeast extract, 0.0625% w/v of cysteine hydrochloride) that differ relative to the kind of vegetable peptone and yeast extract used. Standard culture medium (comprising casein peptone) was used for comparison.
  • Figure 2 Comparison between the bacterial growth (optical density 600 nm) of C .
  • FIG. 21000 Influence on the growth (OD) and enzymatic activity of different concentrations of the yeast extract (0-3% w/v) in the composition of the animal product-free culture medium for C. histolyticum (ATCC 21000) comprising 3% w/v or 4% w/v of soybean peptone and 0.0625% w/v of cysteine hydrochloride.
  • Figure 4 Influence on the growth (OD) and enzymatic activity of different concentrations of the yeast extract (0-3% w/v) in the composition of the animal product-free culture medium for C. histolyticum (ATCC 21000) comprising 3% w/v or 4% w/v of soybean peptone and 0.0625% w/v of cysteine hydrochloride.
  • Figure 4 Influence on the growth (OD) and enzymatic activity of different concentrations of the yeast extract (0-3% w/v) in the composition of the animal product-free culture medium for C. histolyticum (ATCC 21000) comprising 3%
  • AATCC 21000 histolyticum (ATCC 21000) in an animal product-free culture medium comprising: 3% w/v of soybean peptone (NZ SOY BL4), 3% w/v of yeast extract (YE 251) and 0.0625% w/v of cysteine hydrochloride.
  • the bars (1-8) in the chart refer to:
  • Proteinases proteins, peptidases or proteolytic enzymes, EC 3.4
  • proteolytic enzymes are enzymes that cleave peptide bonds between the protein amino acids.
  • Collagen proteins or “collagen” refer to the main structural proteins present in the skin and bones of most animals which are important extracellular matrix components. Collagen molecules consist of 3 chains of individual polypeptide chains (alpha chains) folded in a triple helix configuration that is stabilized by hydrogen bonds.
  • Gellatin refers to a partially hydrolyzed form of collagen.
  • Collagenolytic and gelatinolytic activity refers to the measure of collagen and/or gelatin degradation within a specified period of time. Protease collagenolytic and gelatinolytic activity is commonly verified by the protein's ability to cleave and the degradation rate when in the presence of collagen and gelatins. It is usually expressed in units/mL, units/L, units/mg of enzyme or units/g of enzyme. Collagenolytic and gelatinolytic activity quantification can be determined through a variety of methodologies such as viscometric analysis, colorimetric (ninhydrin; Moore & Stein, JBC, 176, 367, 1948 ) or fluorimetric reactions (collagen and gelatin substrates marked with fluorescence; EnzChek ® ) among others.
  • the activity is quantified following a colorimetric protocol with ninhydrin
  • the activity is measured by the enzymatic hydrolysis of a synthetic substrate (Carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala).
  • Bacterial growth is defined as the division of a bacterial cell in two identical daughter cells during a process called binary fission. This way, the bacterial population duplication occurs at each cell division. However, if the number of viable cells exceeds the average, the bacterial population will present exponential growth. Exponential bacterial growth in a culture can be monitored through well-known methods, such as, direct bacterial cell count (i.e. microscopy, flow cytometry), determining biomass value (milligrams, grams, kilos or tons), colony count, optical density (measured in spectrophotometer, wavelength of about 600nm), monitoring nutrient consumption, among others. Bacterial growth can be characterized by four different phases: “lag phase”, “exponential or log phase", “stationary phase” and "decline or death phase”.
  • the "exponential or log phase” is a period characterized by cell duplication. If growth is not a restraint, cell duplication continues at a steady rate, so both, the number of cells and growth rate, duplicate in each period of time.
  • the exponential growth phase cannot endure indefinitely since the growth medium has nutritional restraints and accumulation of metabolites which are often toxic and are produced by bacterial cells undergoing division.
  • growth rate decreases due to nutritional restraints and accumulation of toxic metabolites. This phase is characterized by resource depletion in the culture medium. In the "decline or death phase", bacteria deplete completely the remaining nutrients still available in the culture medium and die.
  • Pre-inoculum is defined as a suspension of microorganisms obtained from a stock culture that will be used for continued growth of the microorganism for producing of an inoculum.
  • “Inoculum” is a microorganism suspension with a given specific concentration that is to be used for continued growth and/or fermentation on a larger scale (greater volume of culture medium) than the initial one.
  • Anaerobic condition and “anaerobiosis” is defined as the maintenance of a substantially oxygen-free culture condition.
  • “Fresh culture medium” refers to any culture medium for microorganism growth or fermentation that has not been previously used, or, as a medium containing integrally all of its components.
  • Freezing medium characterizes a composition capable of maintaining a microorganism's viability after being subjected to freezing or lyophilization and storage and, thus, its growth and fermentation capacity after the storage period.
  • the storage of the microorganism on the freezing medium can be performed on temperatures that are suitable for its freezing maintenance. Moreover, it is also possible to lyophilize the microorganism in the freezing medium before storage.
  • the freezing medium is an animal product-free and contains, in addition, a cryopreservation agent.
  • cryopreservation agent is a substance intended to protect the microorganism during freeze-thaw allowing it to maintain cell viability after freezing.
  • Some common cryopreservation agents used for microorganism storage are: sucrose, glycerol, dimethyl sulfoxide (DMSO), among others.
  • Wash or storage medium refers to a fraction of microorganism in freezing medium stored for a determined period of time.
  • Supernatant is used hereafter to describe the liquid phase of a microorganism culture medium (growing or fermentation medium), free of particulate or solid material.
  • the supernatant can be obtained by centrifuging and/or filtering the material, such process is well known on the state of the art.
  • Proteins are defined in the present invention as mixtures of compounds yielded by protein hydrolysis (it comprises proteins fragments, whose composition depends on the protein source used for hydrolysis). Commonly, peptones are obtained by acid or enzymatic hydrolysis of natural products, such as animal or vegetable tissues, as well as milk or microbial cultures.
  • the protein source for producing animal origin peptones is often a by-product of meat and dairy production. Therefore, as result of its hydrolysis, there are several other compounds, apart from peptides and amino acids, such as fats, metal ions, salts and vitamins.
  • Animal Product-Free is used to describe a substance completely absence of any animal origin product, component or compound.
  • Animal includes mammals, birds, reptiles, fishes, amphibians, arthropods withal other animal species.
  • Animal excludes microorganisms such as bacteria and yeasts.
  • Animal Product-Free can comprise proteases derived from bacteria of the genus Clostridium, such as C. histolyticum.
  • Animal Product-Free does not include animal-derived proteins, such as: immunoglobulin, meat digests and byproducts, and dairy products and their derivatives.
  • Compound "ingredient”, “product” or “source” “of non-animal origin” are the preferred sources of ingredients for the Clostridium histolyticum culture medium, including vegetables, microorganisms (e.g., yeast) and synthetic compounds.
  • Standard or traditional culture medium refers to the culture medium for growth and/or fermentation of bacteria of the genus Clostridium, in particular C. histolyticum, characterized for comprising animal-origin compounds, including peptone proteose, tryptic digests of casein or meat, casein peptone among others.
  • this invention describes an animal product-free Clostridium culture medium for growth and proteases production possessing collagenolytic and gelatinolytic activity. Most preferably, the present invention describes a culture medium for C. histolyticum.
  • Bacterial growth and protease production with collagenolytic and gelatinolytic activity using an animal product-free culture medium as described in this invention were compared to observations of the same culture parameter with culture medium comprising animal-origin ingredients such as proteose peptone or casein hydrolysate (known as standard or traditional culture medium).
  • animal-origin ingredients such as proteose peptone or casein hydrolysate
  • the culture medium described in the present invention yields a supernatant with higher collagenolytic and gelatinolytic activity than the one obtained from Clostridium histolyticum culture using standard culture medium (comprising animal-origin peptones).
  • this invention presents a culture medium for bacteria of the genus Clostridium, most particularly C. histolyticum, animal product-free medium comprising water, non-animal origin peptone, or its derivatives, yeast extract, cysteine and arginine, or its pharmaceutically acceptable salts.
  • the culture medium of the present invention can additionally comprise one or more additive is selected from the group consisting of agent for adjusting pH within the range of 7.0 and 8.0, reducing agents, inorganic salts, vitamins and mixtures thereof.
  • Optimum levels of growth rate and proteases production presenting collagenolytic and gelatinolytic activity, mainly collagenase, are achieved using the culture medium described on this invention, having a pH ranging from 7.0 to 8.0.
  • the culture medium pH is, preferably, around 7.0. pH adjustment must be performed after sterilization and before initiating C. histolyticum culture , through aqueous solutions of an agent for pH adjustment known by those skilled the art , e.g., ammonium sulphate, NaOH, sulphuric or nitric acid, among others.
  • the additives that can be included on an animal product-free culture medium are selected among substances known by those skilled in the art.
  • the reducing agent is selected from the group consisting of sodium thioglycolate, sodium bisulphite, iron salts, glucose and mixtures thereof.
  • the inorganic salts are selected from the group consisting of NaCl, KCl, Na 2 HPO 4 , NaH 2 PO 4 , KH 2 PO 4 , K 2 HPO 4 , MgSO 4 , FeSO 4 , MnSO 4 and mixtures thereof.
  • Vitamins is selected from the group consisting of biotin, pimelic acid, nicotinamide, calcium pantothenate, folic acid, thiamine nitrate, riboflavin, p-aminobenzoic acid (PABA) and mixtures thereof.
  • Non-animal origin peptones evaluated as ingredients for Clostridium histolyticum culture medium are listed in Table 1. Table 1 .
  • Non-animal origin peptones evaluated as ingredients for Clostridium histolyticum culture medium as described in the present invention Non-animal origin peptones Supplier Amysoy® SheffieldTM Bioscience (Kerry, USA) HYP A® BioSpringer (France) Hy-Pep 1510 ® SheffieldTM Bioscience (Kerry, USA) Hy-Pep 1511 ® SheffieldTM Bioscience (Kerry, USA) Hy-Pep 4601 ® SheffieldTM Bioscience (Kerry, USA) Hy-Pep 4605 ® SheffieldTM Bioscience (Kerry, USA) Hy-Pep 5603 ® SheffieldTM Bioscience (Kerry, USA) Hy-Pep 7504 ® SheffieldTM Bioscience (Kerry, USA) Hy-soy ® SheffieldTM Bioscience (Kerry, USA) NZ-Soy BL4 ® SheffieldTM Bioscience (
  • Non-animal origin peptones can also be obtained from other suppliers, such as: Soy peptone ® (Gibco), Bac-soytone ® (Difco), SE50M ® (DMV), Peptona de soja 312 ® (Biotecnica International, Mexico), SE50MAF-UF ® (DMV), Stedygro Soy ® and Proyield Soy ® .
  • the animal product-free culture medium for Clostridium histolyticum comprising a non-animal origin peptone that is a vegetable peptone selected from the group consisting of soybean, cotton, wheat, sunflower, rice, peanuts, beans, peas, potatoes, sweet corn or a mixture thereof.
  • a vegetable peptone selected from the group consisting of soybean, cotton, wheat, sunflower, rice, peanuts, beans, peas, potatoes, sweet corn or a mixture thereof.
  • the vegetable peptone is a soybean, cotton or wheat peptone, or its mixtures.
  • the vegetable peptone is a soybean peptone.
  • This invention discloses the surprising observation that peptones, or other animal-origin components are not necessary in Clostridium histolyticum culture media, and that the culture medium of the present invention, animal product-free, yields proteases with collagenolytic and gelatinolytic activity superior to that obtained in conventional culture medium containing animal-origin components.
  • vegetable peptones from different suppliers can be used in the animal product-free culture medium for Clostridium histolyticum.
  • the vegetable peptones are hydrolyzed and its production process is also animal product-free.
  • the vegetable peptone is selected from the group consisting of Hy Soy ® , Soytone ® , Amisoy ® , NZ Soy BL4 ® , NZ Soy BL7 ® , Hy Pep 1510 ® , Hy Pep 1511 ® , Hy Pep 5603 ® , Hy Pep 7504 ® , Stedygro Soy ® , SE50M ® , Proyield Soy ® , SE50MAF-UF ® , and mixtures thereof.
  • the vegetable peptone is selected from the group consisting of Hy Pep 4601 ® , NZ soy BL4 ® , HyPep 1511 ® and Hy Pep 7504 ® .
  • the vegetable peptone is NZ soy BL4 ® .
  • the culture medium developed and described in the present invention for Clostridium histolyticum culture contains yeast extract as critical ingredient. Commercial suppliers evaluated for this ingredient are listed in Table 2. Table 2. Yeast extracts evaluated as ingredients for Clostridium histolyticum culture medium described on this invention.
  • the yeast extract is selected from the group consisting of Bacto YE ® , Hy-Pep YE ® , Hy-Yeast 412 ® , Hy-Yeast 413 ® , Hy-Yeast 502 ® , Hy-Yeast 501 ® , Hy-Yeast 503 ® Prodex 710 SD ® , Prodex NS70SD ® , Pronal 5001 ® , YE 11 ® , YE 151 ® , YE 251 ® , YE 70161 ® , YE Y4250 ® , Synth ® , YE 207 ® , YE 5114 ® and mixtures thereof.
  • the yeast extract is YE 251 ® .
  • arginine is L-arginine and cysteine is L-cysteine or its pharmaceutically acceptable salts.
  • the pharmaceutically acceptable salts are cysteine hydrochloride and arginine hydrochloride.
  • the culture medium of the present invention quantitatively, is characterized by comprising non-animal origin peptone at a concentration of 0.5% to 5% w/v; yeast extract present at a concentration of 0.5% to 5% w/v; cysteine, or its pharmaceutically acceptable salts, at a concentration of 0.01% to 0.1% w/v; and arginine, or its pharmaceutically acceptable salts, at a concentration of 0.1% to 1% w/v.
  • the culture medium comprising non-animal origin peptone at a concentration between 1% and 4% w/v, yeast extract at a concentration between 1% and 4% w/v, cysteine, or its pharmaceutically acceptable salts, at a concentration of 0.03% to 0.07% w/v, and arginine, or its pharmaceutically acceptable salts, at a concentration ranging from 0.25% to 0.7% w/v.
  • the culture medium comprising non-animal origin peptone at a concentration of 3% w/v, yeast extract at a concentration of 3% w/v, cysteine, or its pharmaceutically acceptable salts, at a concentration of 0.0625% w/v, and arginine, or its pharmaceutically acceptable salts, at concentration of 0.375% w/v.
  • the culture medium must be sterilized before the inoculum with C. histolyticum.
  • Media sterilization can be performed through methods known by those skilled in the art, for example, treatment by moist heat autoclaving or filtration.
  • the culture medium described in this invention is characterized by being sterile.
  • the animal product-free culture medium of the present invention is characterized by being liquid.
  • the culture medium is used on a process for producing a supernatant from a Clostridium histolyticum liquid culture comprising one or more protease presenting collagenolytic and gelatinolytic activity.
  • C. histolyticum when cultured in the animal product-free liquid medium presented herein, secrete such proteases into the liquid phase.
  • Collagenases, along with other proteases, are the main protein components in the supernatant produced by such C. histolyticum culture.
  • a process for producing supernatant of Clostridium histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activity and which comprising the C. histolyticum culture in an animal product-free culture medium according to the present invention.
  • the process for producing supernatant of Clostridium histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activity comprising the following steps:
  • Anaerobic conditions, as required in steps (b) and (e) can be attained through various methods, including but not limited to the ones described hereafter. Methods for maintaining anaerobic conditions for microorganism growth are well known by those skilled in the art. For instance, the anaerobic conditions in steps (b) and (c), can be maintained through the addition of nitrogen gas to the culture medium during cell growth.
  • culture medium referred in step (a) is to increase cell count from a stock culture, being stored under appropriate conditions for maintaining cell viability.
  • the stock culture is maintained at -80°C in an animal product-free culture medium containing one or more cryopreservation agents.
  • the stock medium is also composed of non-animal derived compounds.
  • the stored stock culture is produced from an animal product-free culture medium comprising vegetable peptones, preferably soybean peptone or its derivatives, yeast extract, cysteine, as well as a cryopreservation agent, in pH between 6.5 and 8.0 and.
  • Cryopreservation agents for microorganisms mainly bacteria, are broadly known by person skilled in the art and can be selected from the group consisting of glycerol, sucrose, dimethyl sulfoxide and glucose.
  • the amount of cryoprotectant added to the stock culture varies depending on the bacterial lineage and must be correctly adjusted prior to preparing the stock microorganism bank (master cell bank and working cell bank).
  • step (b) The purpose of the growth phase (step (b)) is to increase the amount of available and viable microorganisms for production proteases with collagenolytic and gelatinolytic activity, which occurs in step (e). Additionally, the growth phase allows the dormant microorganisms in the stock culture to grow actively and secrete proteases with collagenolytic and gelatinolytic activity at step (e).
  • the sterile and animal product-free culture medium of step (a) of the process described above is characterized by comprising water, vegetable-origin peptone, yeast extract and cysteine.
  • the sterile and animal product-free culture medium of step (a) comprising water, 0.5 to 5% w/v of vegetable peptone, 0.5 to 5% w/v of yeast extract, 0.01 to 0.1% w/v of cysteine or its pharmaceutically acceptable salts and pH of 6.5 to 8.0.
  • the culture medium of step (a) is characterized by comprising water, 3% w/v of vegetable peptone, 3% w/v of yeast extract, 0.0625% w/v of cysteine or its pharmaceutically acceptable salts and pH of 6.5 to 8.0.
  • the preferred culture medium described for step (a) has been defined considering only the minimum composition in order to obtain the presented results, in other words, only the components considered essential for growth.
  • the medium contains, not only the described essential compounds, but also arginine and other additives (inorganic salts, vitamins and reducing agents).
  • the arginine amino acid is essential for the surprising results of the present invention regarding the production of proteases with collagenolytic and gelatinolytic activity (step (e)). It has been observed that the production of proteases with collagenolytic and gelatinolytic activity obtained in a C. histolyticum culture supernatant is much higher when the culture medium contains arginine and cysteine when compared with the non-animal origin medium (only with cysteine) and with the traditional medium containing animal origin compounds.
  • the process for producing supernatant of Clostridium histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activity is characterized by the fact that the sterile animal product-free culture medium described in step (c) comprising water, vegetable peptone, yeast extract, cysteine and arginine, with concentrations defined by the present invention, and a pH of 7.0 to 8.0.
  • the process for producing supernatant of Clostridium histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activity is characterized by the fact that the sterile animal product-free culture medium described in step (c) comprising water, 3% w/v of vegetable peptone, 3% w/v of yeast extract, 0.0625% w/v of cysteine or its pharmaceutically acceptable salts, 0.375% w/v of arginine or its pharmaceutically acceptable salts and a pH between 7.0 and 8.0.
  • the sterile animal product-free culture medium described in step (c) comprising water, 3% w/v of vegetable peptone, 3% w/v of yeast extract, 0.0625% w/v of cysteine or its pharmaceutically acceptable salts, 0.375% w/v of arginine or its pharmaceutically acceptable salts and a pH between 7.0 and 8.0.
  • Bacterial growth can be monitored by measuring the optical density of the culture medium during the process described above (steps (b) and (e)) using a spectrophotometer (reading at 600 nm) according to methods well known by those skilled in the art.
  • the C. histolyticum culture during step (b) may occur in one or more steps.
  • the process for producing supernatant of Clostridium histolyticum liquid culture is characterized by the fact that the step (b) comprising at least the following steps: 1) cultivating the stock culture in a medium culture from step (a) in a ratio of 0.1:1.0 v/v for about 16 hours in order to obtain a pre-inoculum; 2) adding to the pre-inoculum a volume of the culture medium that is equal or superior to the double of the culture medium volume from step (1) and maintaining the culture for about 12 hours in order to obtain an inoculum.
  • step (b) It is essential that the growth during step (b) does not lead to cell lysis before its inoculation into the culture medium in step (c), guaranteeing the cell viability of the inoculum.
  • the process for producing supernatant of Clostridium histolyticum liquid culture can be characterized by comprising only steps from (c) to (f), initiating the C. histolyticum culture directly from a stock culture defined in step (c), without the need of producing an inoculum.
  • the process for producing supernatant of Clostridium histolyticum liquid culture is characterized by the fact that the C. histolyticum culture during step (e) involves the addition, in sufficient amount, of one or more known agents which allow pH adjustment to maintain the pH between 6.5 and 8.0.
  • step (f) of the process for obtaining C. histolyticum collagenolytic enzymes the separation of cellular material or other particulate matter from the culture medium liquid phase can be performed through different techniques of filtration, centrifugation or chromatographic purification known on the state of the art, not limited to tangential filtration, centrifugation, chromatography gel filtration, among others.
  • step (f) is performed through filtration or centrifugation procedures, or both.
  • the process for producing supernatant of Clostridium histolyticum liquid culture is characterized by comprising one or more additional steps for the purification of one or more proteases with collagenolytic and gelatinolytic activity in the supernatant obtained in step (f).
  • Purification can be performed through various filtration methods, chromatographic purification or saline precipitation, not limited to ammonium sulfate precipitation, tangential filtration, ion-exchange chromatography, affinity chromatography, gel filtration chromatography, among others, individually or combined.
  • the supernatant of the C. histolyticum liquid culture is a complex mixture including, not only proteases with collagenolytic and gelatinolytic activity, mainly collagenases, but also other proteins, bacterial metabolites, culture medium components, among other substances.
  • an additional aspect of this invention refers to the supernatant of Clostridium histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activity characterized by being produced through a process described above.
  • the supernatant is characterized by comprising 20 to 90% of collagenases after the purification.
  • the supernatant can be directly purified in order to obtain one or more proteases with collagenolytic and gelatinolytic activity.
  • the supernatant can be stored on a frozen or lyophilized form, without, losing its collagenolytic and gelatinolytic activity.
  • the invention discloses the use of a supernatant of Clostridium histolyticum liquid culture obtained through the process described in the present invention characterized by being for the preparation of a medicament for the treatment of diseases that benefit from collagenolytic and gelatinolytic activity of proteases of C. histolyticum.
  • the invention describes the use of a supernatant of C. histolyticum liquid culture characterized by being for the preparation of a medicament for the treatment of collagen-mediated diseases or diseases due to accumulation of fibrous tissue.
  • the invention describes the use of a supernatant of the C. histolyticum liquid culture characterized by being for the preparation of a medicament for the treatment of necrotic tissue and/or healing processes.
  • a supernatant of the C. histolyticum liquid culture is characterized by being in the preparation of a medicament for the enzymatic debridement.
  • This invention also discloses a pharmaceutical composition characterized by comprising as active ingredient the supernatant of Clostridium histolyticum liquid culture obtained from the process described herein and pharmaceutically acceptable excipients.
  • the pharmaceutical composition can include one or more excipients pharmaceutically acceptable, caps, plugs, carriers, stabilizers, preservatives and thickeners.
  • the pharmaceutical composition, according to the invention is characterized by being for administration in animal or human, for therapeutic or cosmetic use.
  • the pharmaceutical compositions comprising as active ingredient the supernatant of Clostridium histolyticum liquid culture can be administered through oral, sublingual, nasal, intravenous, intramuscular, intraperitoneal, intrarticular, subcutaneous, dermal, transdermal, among others.
  • the pharmaceutical composition, according to the present invention is characterized for being formulated for subcutaneous, dermal and transdermal administration.
  • the pharmaceutical composition is characterized by being for topical use and comprising an activity potency ranging from 0.2 U to 1.8 U of collagenase per gram of composition (quantified through the colorimetric method with ninhydrin).
  • the carriers or pharmaceutically acceptable excipients are selected based on the final composition that may be presented as capsules, tablets, oral solutions, solutions for nasal administration, injectable solution for intramuscular, intravenous, cutaneous or subcutaneous or yet solutions, ointments or creams for topical use, among others.
  • the pharmaceutical compositions of this invention comprises as active ingredient the supernatant of Clostridium histolyticum liquid culture, pharmaceutically acceptable excipients or carriers in the form of a cream or ointment formulation, lyophilized or aqueous solutions.
  • Excipients, carriers or stabilizers pharmaceutically acceptable do not demonstrate toxicity to the recipient organism at the dosages and concentrations used and can include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens such as methyl- and propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; amino acids, monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, sucrose, mannitol, or sorbitol; polysaccharides (such as the polysaccharide hydrophilic Ficoll ® ), polymeric excipient
  • Such pharmaceutical compositions find application in the treatment of diseases that benefit from collagenolytic and gelatinolytic activity of C. histolyticum proteases, in the treatment of pathological conditions of the human body associated with excessive collagen deposition and erratic accumulation of fibrous tissue rich in collagen, treatment of necrotic tissue and healing process and treatment for enzymatic debridement.
  • Examples 1-4 demonstrate the preparation of an animal product-free culture media, according to the present invention, and the C. histolyticum growth and proteases with collagenolytic and gelatinolytic activity production. Results obtained confirm the effectiveness of animal product-free culture medium, related to C. histolyticum growth in comparison with standard culture medium, even without arginine in its composition( Figures 1 and 2 ). The importance of the yeast extract in the medium's composition can be observed in Figure 3 , and the preferred pH range for C. histolyticum growth is exemplified in Figure 4 . Figures 5 and 6 clearly demonstrate that supplementing the animal product-free medium with vitamin and inorganic salts yields no improvement in cell growth nor in protein expression. For such reason such components are considered optional.
  • Example 5 shows the results of culturing in bioreactor (3L) using the animal product-free culture medium described in the present invention.
  • Figure 10 shows the results of a cell culture using animal product-free culture medium without arginine, demonstrating the smaller protease production when compared to one in which arginine was added ( Figure 11 ).
  • Figure 12 shows a comparative graph between animal product-free culture media with and without arginine. The parameters considered in this analysis were bacterial growth rate and enzyme production.
  • Example 6 describes a recovery process to the proteases with collagenolytic and gelatinolytic activity present in the supernatant from the C. histolyticum culture in animal product-free medium described in the present invention.
  • the quality of the supernatant obtained can be highlighted, with no precipitation, facilitating its manipulation.
  • the results of the collagenolytic enzymes recovery by precipitation with ammonium sulfate, followed by dialysis and lyophilization are shown ( Figure 13 ).
  • Example 7 presents a pharmaceutical composition for topic use comprising the supernatant obtained from C. histolyticum liquid culture produced in animal product-free culturemedium.
  • the stability of the compositions is also described; substantiating the quality of the product obtained utilizing the described fermentative processes and defining one more advantageous trait of the present invention.
  • Example 8 describes assays of dermal toxicity with repeated doses (21 days) in rats (male and female), demonstrating the absence of toxicity of the pharmaceutical compositions prepared with collagenolytic enzymes obtained through the lyophilization of the supernatant obtained from C. histolyticum liquid culture produced in animal product-free culture medium, according to the present invention, and through tangential filtration.
  • the absence of pharmaceutical compositions toxicity highlights, once again, the quality of the enzymatic composition of the supernatant from cultures of C. histolyticum in animal product-free culture media, according to the present invention ( Figures 14-17 ).
  • Example 9 shows the results of the cultivation of two C. histolyticum strains, ATCC21000 and T248 in a 3L bioreactor using animal product-free medium as described in the present invention.
  • Figure 14 shows the comparative results, demonstrating an equivalent production of colagenolytic and gelatinolytic proteases by both strains.
  • Example 10 shows the cytotoxicity test results of the colagenolytic and gelatinolytic proteases obtained from the supernatant of the two C. histolyticum strains liquid culture (ATCC 21000 and T248) grown in animal product-free culture medium.
  • C. histolyticum strains may be obtained from several sources, including the American Type of Culture Collections (ATCC). Various Clostridium histolyticum strains have already been applied with the intent of obtaining collagenase; hence the present invention does not limit itself only to the strain utilized for the demonstration through the examples below.
  • ATCC American Type of Culture Collections
  • Clostridium histolyticum strains have already been applied with the intent of obtaining collagenase; hence the present invention does not limit itself only to the strain utilized for the demonstration through the examples below.
  • the ATCC 21000 (non-mobile mutant of a mobile lineage, wild, isolated from the soil ATCC 21000TM) Clostridium histolyticum strain was used in all examples presented herein. The reason for such is due to the fact that this strain is known for yielding high amounts of good quality collagenolytic and gelatinolytic enzymes.
  • the culture medium advised by ATCC for its propagation includes animal origin peptones, inorganic salts and leaver digest (culture medium Reinforced Clostridial Medium - RCM - Oxoid CM149, pH 8.0; anaerobic conditions).
  • Examples 9 and 10 show the results of cultures using animal product-free culture medium as described herein for the T248 Clostridium histolyticum strain, isolated from topsoil sample of a private area (Espirito Santo do Pinhal/SP, Brazil).
  • the T248 strain was characterized morphologically (gram positive bacillus, sporulated), biochemically (optional anaerobic, which degrades gelatins) and genetically ( tpi gene and 16S, 23S ribosomal and 16S and 23S intergenic regions), present exclusively in C. histolyticum strains encoding collagenase class I and II.
  • the strain was also characterized phylogenetically using 16S-23S intergenic region sequences.
  • Example 1 Process for producing standard and animal product-free culture media for Clostridium histolyticum.
  • the standard or traditional culture medium was prepared using the following ingredients, quantities refer per liter of medium: Proteose peptone n. 3 or casein peptone (20 g); yeast extract (5 g); glucose (5 g); sodium thioglycolate (250 mg), pH 7.0 using 5M NaOH.
  • the animal product-free culture medium was prepared using the following ingredients per liter: vegetable origin peptone (10-40 g); yeast extract (0-30 g); cysteine hydrochloride (625 mg), pH 7.0 using 5M NaOH. All components, except glucose which is present only in the standard medium, were dissolved in water and the pH was adjusted to 7.0 using 5M NaOH. The medium was then sterilized in autoclave at 121°C for 20 minutes. A glucose solution was prepared separately and sterilized in an autoclave (autoclave at 121oC for 20 minutes) and this solution was then added to the standard medium only at the time of culture.
  • Example 2 C. histolyticum growth in an animal product-free culture medium and in a standard culture medium as defined in Example 1.
  • the frozen stock culture of Clostridium histolyticum 100 ⁇ L was suspended in 1 mL of standard culture medium or 1 mL of animal product-free culture medium (described in Example 1), separately in two tubes that were filled with the respective culture media to reach the 10 mL final volume (culture denominated as inoculum). Tubes containing the inoculum were incubated at 37 oC, in anaerobiosis, for approximately 16 hours.
  • Figure 1 shows the OD (optical density) results for C. histolyticum (ATCC 21000) growth in an animal product-freeculture medium comprising vegetable peptone (3% w/v) and yeast extract (3% w/v) from different suppliers. The evaluated suppliers display similar results of bacterial growth.
  • Figure 1 it is clearly observed a significant increase in the OD measured when animal product-free culture medium was used, indicating higher bacterial growth rate when compared to the standard medium (with animal origin peptones) (Vetec + Casein). All animal product-free culture media represented in Figure 1 contain 625 mg/L of cysteine hydrochloride and pH of approximately 7.0.
  • Figure 4 represents the results of bacterial growth obtained through the culturing of C. histolyticum in growth medium with pH ranging from 6.5 to 8.5. In the pH range evaluated, only the most basic pH (pH 8.5) negatively influenced the bacterial growth.
  • Example 3 Production of animal product-free culture medium for Clostridium histolyticum and standard culture medium for production of a supernatant comprising proteases with collagenolytic and gelatinolytic activity.
  • the culture medium was prepared according to Example 1.
  • the animal product-free culture medium of the present invention was prepared using the following ingredients per liter: vegetable origin peptone (30 g); yeast extract (30 g); cysteine hydrochloride (0.625 g); arginine hydrochloride (3.75 g) and pH 7.0 (adjusted with 5M NaOH).
  • peptones were evaluated relative to the culturing of C. histolyticum for the production of proteases with collagenolytic and gelatinolytic activity, showing results similar to NZ-Soy BL4 ® .
  • the different yeast extracts evaluated showed similar results to those of the YE 251 ® used in the animal product-free culture media.
  • the vegetable peptones and the yeast extracts suitable to provide the animal product-free culture medium are not limited to the ones used in the examples of this invention. Vegetable peptones and yeast extracts that demonstrate good solubility in water can be used in the preparation of the culture medium for C. histolyticum according to the present invention, since they are animal product-free.
  • the culture medium was sterilized in autoclave at 121 °C for 20 minutes.
  • Example 4 Production of supernatant of Clostridium histolyticum liquid culture comprising one or more proteases with collagenolytic and gelatinolytic activities in an animal product-free culture medium defined in Example 3 and standard culture medium defined in Example 1.
  • Example 3 histolyticum culture in an animal product-free culture medium was carried out as described in Example 2. The culture media evaluated are described in Example 3 (media 1 to 5).
  • the protein profile in the supernatant was evaluated through electrophoresis in 7.5% SDS PAGE under non-reducing conditions (dyed with silver nitrate).
  • the enzymatic activity profile (non-specific) was evaluated in non-reductive SDS-PAGE comprising 10% of gelatin and dyed with Coomassie blue (zymography). Both analytical methodologies are broadly known by those skilled in the art.
  • the collagenolytic and gelatinolytic activity of the obtained supernatants was quantified through EnzChek ® Gelatinase/Collagenase Assay Kit (Molecular Probes, Invitrogen). Assays were carried out in microplates according to manufacturer instructions, using gelatin and/or collagen fluoresceine conjugated as substrate. Fluorescence determinations were made using a fluorescence reader. Briefly, 100 ⁇ L of the culture supernatant, after centrifugation, were mixed to the substrate diluted in the incubation buffer prepared according to the manufacturer instructions. Samples were incubated at 37 oC for 15 minutes and the fluorescence was determined at 515 nm using a fluorescence microplate reader. As a negative control sterile culture medium was used instead of the culture supernatant. As a positive control, it was used a Collagenase reagent provided with the kit and prepared according to manufacturer instructions.
  • Bacterial growth was determined by optical density - OD - using a spectrophotometer at 600 nm.
  • the animal product-free culture medium supplemented (medium 2; comprising vegetable peptone, yeast extract and cysteine) with vitamins, inorganic salts and amino acids was evaluated.
  • This experiment demonstrated a small influence of these additives in cell growth as well as protease expression with collagenolytic and gelatinolytic activities from supernatant of Clostridium histolyticum liquid culture ( Figure 5 ).
  • Medium 1 was used as a control for this assay.
  • Figure 8 demonstrates that the C. histolyticum culture in an animal product-free culture medium as described in the present invention, comprising amino acids cysteine and arginine (medium 4), results surprisingly in a supernatant with significantly higher collagenolytic and gelatinolytic activity than the supernatant obtained when using animal product-free culture medium containing only the amino acid cysteine (Medium 1, used as a control of this assay).
  • Such effect is due to arginine since the addition of another amino acid, for instance glycine, leads to reduction of collagenolytic and gelatinolytic activity observed.
  • Figure 9 shows results of collagenolytic and gelatinolytic activity and OD (600 nm) observed in C. histolyticum culture in medium 3 (animal product-free culture medium comprising cysteine and amino acid groups).
  • a culture using medium 1 was carried out (animal product-free culture medium containing only the amino acid cysteine). It can be observed that no amino acid or amino acid combination added to the culture medium was able to increase collagenolytic and gelatinolytic activity, which was surprisingly observed in the supernatant from C. histolyticum liquid culture comprising amino acid arginine ( Figure 8 ).
  • Example 5 Production of a Clostridium histolyticum liquid culture comprising proteases with collagenolytic and gelatinolytic activities in an animal product-free culture medium - bioreactors culture.
  • the frozen stock culture of Clostridium histolyticum (100 ⁇ L) was suspended in 1 mL animal product-free culture medium (medium 1, as defined below), and then the culture medium was added to final volume of 10 mL (culture denominated as pre-inoculum). Tubes containing the pre-inoculums were incubated at 37oC under anaerobic conditions for approximately 16 hours.After this period, 3 mL of the aforesaid pre-inoculum was then incubated in 250 mL flasks, containing 150 mL of the respective C. histolyticum culture medium. This was denominated inoculum, and it was maintained at 37 oC during 12-14 hours under anaerobic conditions (attained through the addition of nitrogen gas).
  • 1 - Cell bank preparation a cell bank was prepared using ATCC 21000 strain. It was grown in agar plates in which the medium was composed of: soy peptone, yeast extract and agar. Colonies with apparent higher cell growth were isolated and inoculated in cell bank medium containing the same ingredients as the solid medium, with the exception of agar. In order to preserve the cells during freeze and thaw, thus maintaining its viability, 10% sterile glycerol was added to the C. histolyticum culture derived from the isolated colonies. This composition was then fractionated into cryotubes and frozen at -80 oC for future use, such conditions allows the stock cultures to be used for at least 1 year without losing cell viability.
  • pre-inoculum and inoculum preparation a cell bank aliquot was rapidly thawed at room temperature ; a 100 ⁇ L aliquot was transferred to 1 mL of an animal product-free culture medium (Medium 1), to which 9 mL of the same medium was added for bacterial growth at 37 oC under anaerobic conditions for about 16 hours (pre-inoculum). Subsequently, the pre-inoculums were used to generate the inoculums as described previously.
  • Medium 1 animal product-free culture medium
  • 3 - Fermentation phase production of supernatant of C . histolyticum liquid culture comprising proteases with collagenolytic and gelatinolytic activities
  • 60 mL of the inoculum were transferred to a fermenter containing 3 L of animal product-free culture medium (pH 7.0, Medium 1) and maintained at 37 oC for about 14 hours under anaerobic conditions, through addition of nitrogen gas.
  • the pH was controlled via the addition of 5M NH 4 OH when the pH reached values lower than 6.5, or 2MH 2 SO 4 when the pH reached values higher than 8.0.
  • Bacterial growth was monitored through optical density measurements (600 nm) which were performed using a spectrophotometer every 2 hours of culturing.
  • the protein and enzymatic supernatant profile obtained from the C. histolyticum cultures described above were evaluated through gel electrophoresis (7.5% SDS PAGE) under non-reducing conditions and dyed with silver nitrate ( Figure 11 ) and non-reducing SDS-PAGE comprising 10% gelatin and dyed with Coomassie blue ( Figures 10 and 11 ; zymography).
  • Figure 11 shows the results (OD, enzymatic activity, SDS-PAGE GEL/silver nitrate and zymography) of a fermentative process carried out in Medium 6. It can be observed in Figure 11 , a bacterial growth curve similar to that obtained when the fermentation took place in a bioreactor using medium 1 (comprising cysteine only, Figure 10 ). However and surprisingly, it can also be observed a significantly higher collagenolytic and gelatinolytic activity, showing that the addition of arginine to the animal product-free culture medium influences the increase of collagenolytic and gelatinolytic enzymes production in C. histolyticum cultures (activity in U/mL).
  • Figure 12 shows the comparison of collagenolytic and gelatinolytic activity (mU/mL) and optical density (OD 600 nm) between the culture using Medium 1 (containing only amino acid cysteine) and the culture using Medium 6 (containing cysteine and arginine), clearly showing an increase on average of 30% of the collagenolytic and gelatinolytic activity yielded in a C. histolyticum culture supernatant. Additionally, images of SDS-PAGE dyed with silver nitrate and zymography of the supernatants obtained from the C. histolyticum cultures are presented in Figures 10 and 11 , portraying the supernatant quality, predominantly having proteins with high molecular weight, identified here as being collagenases.
  • Example 6 Recovery of proteases with collagenolytic and gelatinolytic activity obtained from a C. histolyticum liquid culture supernatant when cultured in an animal product-free culture medium.
  • the supernatant comprising the proteases with collagenolytic and gelatinolytic activity was obtained through the C. histolyticum culture according to Example 5, using Medium 6 (comprising cysteine and arginine) in step 3.
  • Example 7 Pharmaceutical composition for topical use comprising C. histolyticum collagenases - Stability Assays
  • the pharmaceutical composition used to exemplify the present invention was a dermatological ointment comprising the ingredients presented below: Quantity/ g Ingredient Formulation 1 Formulation 2 Formulation 3 Collagenase 0.2 U 0.6 U 1.8 U Liquid vaseline - mineral oil 0.0188 mL 0.0188 mL 0.0188 mL Solid vaseline - MBK 0.9846 g 0.9846 g 0.9846 g
  • the specific collagenase activity was determined by enzymatic hydrolysis of a synthetic substrate Carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala, being classified through colorimetric assay with ninhydrin.
  • An enzyme unit is defined as being responsible for the release of 1 ⁇ mol of Gly-Pro-Ala from Z-Gly-Pro-Gly-Gly-Pro-Ala hexapeptide in 1 minute at 37oC.
  • the pharmaceutical composition was prepared through the dispersion of the lyophilized component (amount equivalent to the specific collagenase activity equal to 0.2 U, 0.6 U and 1.8 U/g of formulation), obtained according to Example 5 (lyophilized component of the supernatant obtained through a culture using Medium 6 after tangential flow filtration), in the other excipients and homogenized.
  • the 0.6 U/g dose is considered a therapeutic dose proper for human use.
  • Example 8 Pharmaceutical composition for topic use comprising C . histolyticum collagenases - Toxicity
  • compositions used to exemplify the present invention are the same used to perform stability assays described above (Example 7).
  • the assay based on the OECD 410 Guideline, consisted in surgically provoking an injury using a 4mm sterile punch at the center of the dorsal median line on the first day of application (dorsal area hair removed by an epilator device).
  • a topical application of the composition (0.425g; compositions containing 0.2 U/g, 0.6 U/g and 1.8 U/g of collagenase), to an area equivalent to 30-40 cm 2 , was done with a spatula, inside and around the injury, once-a-day, for 21 days. This was performed in three groups of Rattus norvegicus (albino variety, Wistar lineage) in which there were 10 rats per group, 5 males and 5 nulliparous and non-pregnant females.
  • the application site was preserved with gauze and a nonirritating tape for about 24 hours after the application.
  • the obtained results demonstrated that the application of the pharmaceutical composition comprising the lyophilized component (amount equivalent to the specific collagenase activity equal to 0.2 U, 0.6 U and 1.8 U/g formulation), obtained according to Example 5 (supernatant lyophilized component obtained after tangential flow filtration procedure) over a period of 21 days via dermal exposure to Rattus novergicus in the 0.2 U/g, 0.6 U/g and 1.8 U/g formulations, was well tolerated and did not provoke changes possibly related to toxicity. It may surprisingly be observed that even with a dose therapeutic three-times superior than that considered for humans, the described composition in the present invention does not show signs of toxicity in rats. Table 5. Body weight (male and female rats).
  • Trial groups for the evaluation of dermal toxicity through repeated doses (21 days) of topical composition comprising purified supernatant and lyophilized component derived from C. histolyticum culture (ATCC 21000) using an animal product-free culture medium comprising soy peptone (30 g/L), yeast extract (30 g/L), cysteine hydrochloride (0.625 g/L) and arginine hydrochloride (3.75 g/L).
  • Control composition only with excipients.
  • Example 9 Production of supernatant of C. histolyticum liquid culture (ATCC 21000 and T248 strains) comprising proteases with collagenolytic and gelatinolytic activities in animal product-free culture medium - bioreactors culture.
  • Figure 14 shows a comparative analysis of the collagenolytic and gelatinolytic activities (mU/mL) and optical density (OD 600 nm) between Clostridium histolyticum ATCC 21000 and T248 cultures. There is no significant difference (statistical analysis: Mann Whitney, p ⁇ 0.05) between the collagenolytic and gelatinolytic activity of the supernatants obtained from the culture of both C. histolyticum strains.
  • Example 10 Cytotoxicity test of collagenolytic and gelatinolytic proteases obtained from Clostridium histolyticum (ATCC 21000 e T248) liquid culture supernatant using an animal product-free culture medium.
  • the collagenolytic and gelatinolytic proteases were obtained according to Example 6 (lyophilize samples).
  • the cells used in the cytotoxicity test were of fibroblastic type, from a lineage established in V79 clone M-8 cultures from Chinese Hamster ( Cricetulus griseus ) lung.
  • the fibroblasts were maintained in 25cm 2 culture flasks (TPP, Techno Plastic Products AG, Trasadingen, Switzerland) until the confluence density was achieved.
  • the culture was carried out (performed) in DMEM culture medium supplemented with 10% of bovine calf serum (Nutricell), 100 UI/mL of penicillin and 100 ⁇ g/mL of streptomycin sulfate(Nutricell), in a 37°C incubator with humidified atmosphere with 5% CO 2 (Melo et al., 2000, 2001).
  • the cytotoxicity test was performed in 96 well plates (IWAKI, Asahi Techno Glass, Co., Funabasi, Japan), with 3 x 10 4 cells/mL in each well, followed by a 48 hour incubation at 37°C period. The initial cellular viability was verified through a Trypan Blue exclusion test. After the incubation period of 48 hours the medium was replaced by a supplemented DMEM medium with different concentrations (up to 1 mg/mL) of the freeze-dried samples, described below: Sample Origin Strains 1 acquired from third-parties - 2 acquired from third-parties - 3 Obtained according to the present invention (Example 6) ATCC 21000 4 Obtained according to the present invention (Example 6) ATCC 21000
  • the collagenolytic and gelatinolytic proteases obtained from C. histolyticum cultures according to the present invention did not demonstrate cytotoxicity, as observed in the results presented in Table 9.
  • Table 9 Cytotoxicity test results of collagenolytic and gelatinolytic proteases obtained from C. histolyticum cultures, ATCC 21000 e T248, supernatants produced according to the present invention, and collagenolytic and gelatinolytic proteases present in the raw material acquired from third parties.

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WO2016175565A1 (fr) 2015-04-28 2016-11-03 Daewoong Co., Ltd. Composition de milieu pour la préparation de toxine botulinique
WO2017074257A1 (fr) * 2015-10-26 2017-05-04 Nanyang Technological University Dérivés non viables de clostridium sporogenes utilisés comme agents thérapeutiques anti-cancéreux
WO2023131588A1 (fr) 2022-01-05 2023-07-13 Nordmark Pharma Gmbh Milieu de culture pour la culture de hathewaya histolytica (ou clostridium histolyticum) et la production d'une ou de plusieurs protéases

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EP4013851A1 (fr) * 2019-08-16 2022-06-22 Janssen Biotech, Inc. Milieux de culture optimisés pour bactéries clostridia
CN113481120B (zh) * 2021-06-29 2023-03-21 广州知易生物科技有限公司 培养基及其制备方法、用其培养脆弱拟杆菌的方法

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WO2016175565A1 (fr) 2015-04-28 2016-11-03 Daewoong Co., Ltd. Composition de milieu pour la préparation de toxine botulinique
EP3289069A4 (fr) * 2015-04-28 2018-10-03 Daewoong Co., Ltd. Composition de milieu pour la préparation de toxine botulinique
US10465179B2 (en) 2015-04-28 2019-11-05 Daewoong Co., Ltd. Medium composition for preparing botulinum toxin
WO2017074257A1 (fr) * 2015-10-26 2017-05-04 Nanyang Technological University Dérivés non viables de clostridium sporogenes utilisés comme agents thérapeutiques anti-cancéreux
WO2023131588A1 (fr) 2022-01-05 2023-07-13 Nordmark Pharma Gmbh Milieu de culture pour la culture de hathewaya histolytica (ou clostridium histolyticum) et la production d'une ou de plusieurs protéases

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